Publications by authors named "Guido Mastrobuoni"

41 Publications

Rbm10 facilitates heterochromatin assembly via the Clr6 HDAC complex.

Epigenetics Chromatin 2021 Jan 19;14(1). Epub 2021 Jan 19.

Department of Biology, New York University, New York, NY, 10003-6688, USA.

Splicing factors have recently been shown to be involved in heterochromatin formation, but their role in controlling heterochromatin structure and function remains poorly understood. In this study, we identified a fission yeast homologue of human splicing factor RBM10, which has been linked to TARP syndrome. Overexpression of Rbm10 in fission yeast leads to strong global intron retention. Rbm10 also interacts with splicing factors in a pattern resembling that of human RBM10, suggesting that the function of Rbm10 as a splicing regulator is conserved. Surprisingly, our deep-sequencing data showed that deletion of Rbm10 caused only minor effect on genome-wide gene expression and splicing. However, the mutant displays severe heterochromatin defects. Further analyses indicated that the heterochromatin defects in the mutant did not result from mis-splicing of heterochromatin factors. Our proteomic data revealed that Rbm10 associates with the histone deacetylase Clr6 complex and chromatin remodelers known to be important for heterochromatin silencing. Deletion of Rbm10 results in significant reduction of Clr6 in heterochromatin. Our work together with previous findings further suggests that different splicing subunits may play distinct roles in heterochromatin regulation.
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http://dx.doi.org/10.1186/s13072-021-00382-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816512PMC
January 2021

Inhibiting phosphoglycerate dehydrogenase counteracts chemotherapeutic efficacy against MYCN-amplified neuroblastoma.

Int J Cancer 2021 Mar 17;148(5):1219-1232. Epub 2020 Dec 17.

Department of Pediatric Hematology and Oncology, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany.

Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.
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http://dx.doi.org/10.1002/ijc.33423DOI Listing
March 2021

Integrative functional genomics decodes herpes simplex virus 1.

Nat Commun 2020 04 27;11(1):2038. Epub 2020 Apr 27.

Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Versbacher Straße 7, 97078, Würzburg, Germany.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.
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http://dx.doi.org/10.1038/s41467-020-15992-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184758PMC
April 2020

Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription.

Mol Cell 2020 03 31;77(6):1322-1339.e11. Epub 2020 Jan 31.

Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany. Electronic address:

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.
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http://dx.doi.org/10.1016/j.molcel.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086158PMC
March 2020

Kinetic modelling of quantitative proteome data predicts metabolic reprogramming of liver cancer.

Br J Cancer 2020 01 10;122(2):233-244. Epub 2019 Dec 10.

Molecular Tumor Biology, Department of General, Visceral and Transplantation Surgery, RWTH University Hospital, 52074, Aachen, Germany.

Background: Metabolic alterations can serve as targets for diagnosis and cancer therapy. Due to the highly complex regulation of cellular metabolism, definite identification of metabolic pathway alterations remains challenging and requires sophisticated experimentation.

Methods: We applied a comprehensive kinetic model of the central carbon metabolism (CCM) to characterise metabolic reprogramming in murine liver cancer.

Results: We show that relative differences of protein abundances of metabolic enzymes obtained by mass spectrometry can be used to assess their maximal velocity values. Model simulations predicted tumour-specific alterations of various components of the CCM, a selected number of which were subsequently verified by in vitro and in vivo experiments. Furthermore, we demonstrate the ability of the kinetic model to identify metabolic pathways whose inhibition results in selective tumour cell killing.

Conclusions: Our systems biology approach establishes that combining cellular experimentation with computer simulations of physiology-based metabolic models enables a comprehensive understanding of deregulated energetics in cancer. We propose that modelling proteomics data from human HCC with our approach will enable an individualised metabolic profiling of tumours and predictions of the efficacy of drug therapies targeting specific metabolic pathways.
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http://dx.doi.org/10.1038/s41416-019-0659-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052204PMC
January 2020

Context-specific regulation of cell survival by a miRNA-controlled BIM rheostat.

Genes Dev 2019 12 7;33(23-24):1673-1687. Epub 2019 Nov 7.

Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch 13125, Germany.

Knockout of the ubiquitously expressed miRNA-17∼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17∼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17∼92:Bim interactions to the complex miR-17∼92 knockout phenotype, we used a system of conditional mutagenesis of the nine 3' UTR miR-17∼92 seed matches. Blocking miR-17∼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17∼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by haploinsufficiency. Thus, the interaction of miR-17∼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.
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http://dx.doi.org/10.1101/gad.330134.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942046PMC
December 2019

C/EBPβ-LIP induces cancer-type metabolic reprogramming by regulating the /LIN28B circuit in mice.

Commun Biol 2019 14;2:208. Epub 2019 Jun 14.

1European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen, University of Groningen, 9700 AD Groningen, The Netherlands.

The transcription factors LAP1, LAP2 and LIP are derived from the -mRNA through the use of alternative start codons. High LIP expression has been associated with human cancer and increased cancer incidence in mice. However, how LIP contributes to cellular transformation is poorly understood. Here we present that LIP induces aerobic glycolysis and mitochondrial respiration reminiscent of cancer metabolism. We show that LIP-induced metabolic programming is dependent on the RNA-binding protein LIN28B, a translational regulator of glycolytic and mitochondrial enzymes with known oncogenic function. LIP activates LIN28B through repression of the microRNA family that targets the -mRNA. Transgenic mice overexpressing LIP have reduced levels of and increased LIN28B expression, which is associated with metabolic reprogramming as shown in primary bone marrow cells, and with hyperplasia in the skin. This study establishes LIP as an inducer of cancer-type metabolic reprogramming and as a regulator of the /LIN28B regulatory circuit.
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http://dx.doi.org/10.1038/s42003-019-0461-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572810PMC
May 2020

Alterations of mTOR signaling impact metabolic stress resistance in colorectal carcinomas with BRAF and KRAS mutations.

Sci Rep 2018 06 15;8(1):9204. Epub 2018 Jun 15.

Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin Institute of Health (BIH), Robert-Roessle-Str. 10, 13125, Berlin, Germany.

Metabolic reprogramming is as a hallmark of cancer, and several studies have reported that BRAF and KRAS tumors may be accompanied by a deregulation of cellular metabolism. We investigated how BRAF and KRAS affect cell metabolism, stress resistance and signaling in colorectal carcinoma cells driven by these mutations. KRAS expressing cells are characterized by the induction of glycolysis, accumulation of lactic acid and sensitivity to glycolytic inhibition. Notably mathematical modelling confirmed the critical role of MCT1 designating the survival of KRAS cells. Carcinoma cells harboring BRAF remain resistant towards alterations of glucose supply or application of signaling or metabolic inhibitors. Altogether these data demonstrate that an oncogene-specific decoupling of mTOR from AMPK or AKT signaling accounts for alterations of resistance mechanisms and metabolic phenotypes. Indeed the inhibition of mTOR in BRAF cells counteracts the metabolic predisposition and demonstrates mTOR as a potential target in BRAF-driven colorectal carcinomas.
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http://dx.doi.org/10.1038/s41598-018-27394-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003911PMC
June 2018

Proteomic analysis of chemosensory organs in the honey bee parasite Varroa destructor: A comprehensive examination of the potential carriers for semiochemicals.

J Proteomics 2018 06 10;181:131-141. Epub 2018 Apr 10.

Biology Department, University of Firenze, via Madonna del Piano 6, 50019 Sesto Fiorentino, Italy. Electronic address:

We have performed a proteomic analysis on chemosensory organs of Varroa destructor, the honey bee mite, in order to identify putative soluble carriers for pheromones and other olfactory cues emitted by the host. In particular, we have analysed forelegs, mouthparts (palps, chelicera and hypostome) and the second pair of legs (as control tissue) in reproductive and phoretic stages of the Varroa life cycle. We identified 958 Varroa proteins, most of them common to the different organs and stages. Sequence analysis shows that four proteins can be assigned to the odorant-binding protein (OBP)-like class, which bear some similarity to insect OBPs, but so far have only been reported in some Chelicerata. In addition, we have detected the presence of two proteins belonging to the Niemann-Pick family, type C2 (NPC2), which have also been suggested as semiochemical carriers. Biological significance: The mite Varroa destructor is the major parasite of the honey bee and is responsible for great economical losses. The biochemical tools used by Varroa to detect semiochemicals produced by the host are still largely unknown. This work contributes to understand the molecular basis of olfaction in Varroa and, more generally, how detection of semiochemicals has evolved in terrestrial non-hexapod Arthropoda. Moreover, the identification of molecular carriers involved in olfaction can contribute to the development of control strategies for this important parasite.
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http://dx.doi.org/10.1016/j.jprot.2018.04.009DOI Listing
June 2018

The mRNA 3'-UTR couples RNA polymerase II function to glutamine and ribonucleotide levels.

EMBO J 2017 07 13;36(13):1854-1868. Epub 2017 Apr 13.

Theodor Boveri Institute and Comprehensive Cancer Center Mainfranken, Biocenter, University of Würzburg, Würzburg, Germany

Deregulated expression of enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous via the 3'-UTR of the mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.
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http://dx.doi.org/10.15252/embj.201796662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494468PMC
July 2017

On Mass Ambiguities in High-Resolution Shotgun Lipidomics.

Anal Chem 2017 03 21;89(5):2986-2994. Epub 2017 Feb 21.

Berlin Institute of Health Technology Platform Metabolomics, Max-Delbrück-Centrum for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin-Buch, Germany.

Mass-spectrometry-based lipidomics aims to identify as many lipid species as possible from complex biological samples. Due to the large combinatorial search space, unambiguous identification of lipid species is far from trivial. Mass ambiguities are common in direct-injection shotgun experiments, where an orthogonal separation (e.g., liquid chromatography) is missing. Using the rich information within available lipid databases, we generated a comprehensive rule set describing mass ambiguities, while taking into consideration the resolving power (and its decay) of different mass analyzers. Importantly, common adduct species and isotopic peaks are accounted for and are shown to play a major role, both for perfect mass overlaps due to identical sum formulas and resolvable mass overlaps. We identified known and hitherto unknown mass ambiguities in high- and ultrahigh resolution data, while also ranking lipid classes by their propensity to cause ambiguities. On the basis of this new set of ambiguity rules, guidelines and recommendations for experimentalists and software developers of what constitutes a solid lipid identification in both MS and MS/MS were suggested. For researchers new to the field, our results are a compact source of ambiguities which should be accounted for. These new findings also have implications for the selection of internal standards, peaks used for internal mass calibration, optimal choice of instrument resolution, and sample preparation, for example, in regard to adduct ion formation.
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http://dx.doi.org/10.1021/acs.analchem.6b04456DOI Listing
March 2017

Proteomics Quality Control: Quality Control Software for MaxQuant Results.

J Proteome Res 2016 Mar 28;15(3):777-87. Epub 2015 Dec 28.

Max-Delbrück-Centrum for Molecular Medicine Berlin , Robert-Rössle-Straße 10, 13125 Berlin, Germany.

Mass spectrometry-based proteomics coupled to liquid chromatography has matured into an automatized, high-throughput technology, producing data on the scale of multiple gigabytes per instrument per day. Consequently, an automated quality control (QC) and quality analysis (QA) capable of detecting measurement bias, verifying consistency, and avoiding propagation of error is paramount for instrument operators and scientists in charge of downstream analysis. We have developed an R-based QC pipeline called Proteomics Quality Control (PTXQC) for bottom-up LC-MS data generated by the MaxQuant software pipeline. PTXQC creates a QC report containing a comprehensive and powerful set of QC metrics, augmented with automated scoring functions. The automated scores are collated to create an overview heatmap at the beginning of the report, giving valuable guidance also to nonspecialists. Our software supports a wide range of experimental designs, including stable isotope labeling by amino acids in cell culture (SILAC), tandem mass tags (TMT), and label-free data. Furthermore, we introduce new metrics to score MaxQuant's Match-between-runs (MBR) functionality by which peptide identifications can be transferred across Raw files based on accurate retention time and m/z. Last but not least, PTXQC is easy to install and use and represents the first QC software capable of processing MaxQuant result tables. PTXQC is freely available at https://github.com/cbielow/PTXQC .
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http://dx.doi.org/10.1021/acs.jproteome.5b00780DOI Listing
March 2016

Extensive identification and analysis of conserved small ORFs in animals.

Genome Biol 2015 Sep 14;16:179. Epub 2015 Sep 14.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany.

Background: There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging.

Results: Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates.

Conclusions: Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo.
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http://dx.doi.org/10.1186/s13059-015-0742-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568590PMC
September 2015

RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-κB pathway.

Nat Commun 2015 Jul 14;6:7367. Epub 2015 Jul 14.

RNA Biology and Posttranscriptional Regulation, Berlin Institute of Medical Systems Biology at the Max-Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFα mRNA decay via a 3'UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify ∼ 3,800 mRNA targets with >16,000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-κB pathway regulators such as IκBα and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3'UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IκB kinase and NF-κB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-κB pathway.
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http://dx.doi.org/10.1038/ncomms8367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510711PMC
July 2015

MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.

Mol Cell 2014 May 10;54(4):573-85. Epub 2014 Apr 10.

Max-Delbrück-Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, 13125 Berlin, Germany. Electronic address:

RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs.
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http://dx.doi.org/10.1016/j.molcel.2014.03.017DOI Listing
May 2014

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae.

PLoS One 2013 25;8(11):e75162. Epub 2013 Nov 25.

Integrative Proteomics and Metabolomics, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075162PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839933PMC
January 2015

Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation.

RNA Biol 2013 Jul 29;10(7):1146-59. Epub 2013 May 29.

Systems Biology of Gene Regulatory Elements; Max-Delbrück-Center for Molecular Medicine; Berlin, Germany.

The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA-binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B-binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28-binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knockdown, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts.
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http://dx.doi.org/10.4161/rna.25194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849162PMC
July 2013

The pro-neurotrophin receptor sortilin is a major neuronal apolipoprotein E receptor for catabolism of amyloid-β peptide in the brain.

J Neurosci 2013 Jan;33(1):358-70

Max Delbrueck Center for Molecular Medicine, D-13125 Berlin, Germany.

Apolipoprotein E (APOE) is the major risk factor for sporadic Alzheimer's disease. Among other functions, APOE is proposed to sequester neurotoxic amyloid-β (Aβ) peptides in the brain, delivering them to cellular catabolism via neuronal APOE receptors. Still, the receptors involved in this process remain controversial. Here, we identified the pro-neurotrophin receptor sortilin as major endocytic pathway for clearance of APOE/Aβ complexes in neurons. Sortilin binds APOE with high affinity. Lack of receptor expression in mice results in accumulation of APOE and of Aβ in the brain and in aggravated plaque burden. Also, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aβ complexes despite proper expression of other APOE receptors. Despite higher than normal brain APOE levels, sortilin-deficient animals display anomalies in brain lipid metabolism (e.g., accumulation of sulfatides) seen in APOE-deficient mice, indicating functional deficiency in cellular APOE uptake pathways. Together, our findings identified sortilin as an essential neuronal pathway for APOE-containing lipoproteins in vivo and suggest an intriguing link between Aβ catabolism and pro-neurotrophin signaling converging on this receptor.
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http://dx.doi.org/10.1523/JNEUROSCI.2425-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744345PMC
January 2013

Proteome dynamics and early salt stress response of the photosynthetic organism Chlamydomonas reinhardtii.

BMC Genomics 2012 May 31;13:215. Epub 2012 May 31.

Max Delbrück Center for Molecular Medicine Berlin, Berlin Institute for Medical Systems Biology (BIMSB), Berlin, Germany.

Background: The cellular proteome and metabolome are underlying dynamic regulation allowing rapid adaptation to changes in the environment. System-wide analysis of these dynamics will provide novel insights into mechanisms of stress adaptation for higher photosynthetic organisms. We applied pulsed-SILAC labeling to a photosynthetic organism for the first time and we established a method to study proteome dynamics in the green alga Chlamydomonas reinhardtii, an emerging model system for plant biology. In addition, we combined the analysis of protein synthesis with metabolic profiling to study the dynamic changes of metabolism and proteome turnover under salt stress conditions.

Results: To study de novo protein synthesis an arginine auxotroph Chlamydomonas strain was cultivated in presence of stable isotope-labeled arginine for 24 hours. From the time course experiment in 3 salt concentrations we could identify more than 2500 proteins and their H/L ratio in at least one experimental condition; for 998 protiens at least 3 ratio counts were detected in the 24 h time point (0 mM NaCl). After fractionation we could identify 3115 proteins and for 1765 of them we determined their de novo synthesis rate. Consistently with previous findings we showed that RuBisCO is among the most prominent proteins in the cell; and similar abundance and turnover for the small and large RuBisCO subunit could be calculated. The D1 protein was identified among proteins with a high synthesis rates. A global median half-life of 45 h was calculated for Chlamydomonas proteins under the chosen conditions.

Conclusion: To investigate the temporal co-regulation of the proteome and metabolome, we applied salt stress to Chlamydomonas and studied the time dependent regulation of protein expression and changes in the metabolome. The main metabolic response to salt stress was observed within the amino acid metabolism. In particular, proline was up-regulated manifold and according to that an increased carbon flow within the proline biosynthetic pathway could be measured. In parallel the analysis of abundance and de novo synthesis of the corresponding enzymes revealed that metabolic rearrangements precede adjustments of protein abundance.
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http://dx.doi.org/10.1186/1471-2164-13-215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444938PMC
May 2012

Gene expression of pluripotency determinants is conserved between mammalian and planarian stem cells.

EMBO J 2012 Jun 27;31(12):2755-69. Epub 2012 Apr 27.

Laboratory of Systems Biology of Gene Regulatory Elements, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

Freshwater planaria possess extreme regeneration capabilities mediated by abundant, pluripotent stem cells (neoblasts) in adult animals. Although planaria emerged as an attractive in vivo model system for stem cell biology, gene expression in neoblasts has not been profiled comprehensively and it is unknown how molecular mechanisms for pluripotency in neoblasts relate to those in mammalian embryonic stem cells (ESCs). We purified neoblasts and quantified mRNA and protein expression by sequencing and shotgun proteomics. We identified ∼4000 genes specifically expressed in neoblasts, including all ∼30 known neoblast markers. Genes important for pluripotency in ESCs, including regulators as well as targets of OCT4, were well conserved and upregulated in neoblasts. We found conserved expression of epigenetic regulators and demonstrated their requirement for planarian regeneration by knockdown experiments. Post-transcriptional regulatory genes characteristic for germ cells were also enriched in neoblasts, suggesting the existence of a common ancestral state of germ cells and ESCs. We conclude that molecular determinants of pluripotency are conserved throughout evolution and that planaria are an informative model system for human stem cell biology.
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http://dx.doi.org/10.1038/emboj.2012.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380209PMC
June 2012

In vivo and transcriptome-wide identification of RNA binding protein target sites.

Mol Cell 2011 Dec;44(5):828-40

Systems Biology of Gene Regulatory Elements, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes.
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http://dx.doi.org/10.1016/j.molcel.2011.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253457PMC
December 2011

De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics.

Genome Res 2011 Jul 2;21(7):1193-200. Epub 2011 May 2.

Max-Delbrück-Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, Robert Rössle Strasse 10, Berlin, Germany.

Freshwater planaria are a very attractive model system for stem cell biology, tissue homeostasis, and regeneration. The genome of the planarian Schmidtea mediterranea has recently been sequenced and is estimated to contain >20,000 protein-encoding genes. However, the characterization of its transcriptome is far from complete. Furthermore, not a single proteome of the entire phylum has been assayed on a genome-wide level. We devised an efficient sequencing strategy that allowed us to de novo assemble a major fraction of the S. mediterranea transcriptome. We then used independent assays and massive shotgun proteomics to validate the authenticity of transcripts. In total, our de novo assembly yielded 18,619 candidate transcripts with a mean length of 1118 nt after filtering. A total of 17,564 candidate transcripts could be mapped to 15,284 distinct loci on the current genome reference sequence. RACE confirmed complete or almost complete 5' and 3' ends for 22/24 transcripts. The frequencies of frame shifts, fusion, and fission events in the assembled transcripts were computationally estimated to be 4.2%-13%, 0%-3.7%, and 2.6%, respectively. Our shotgun proteomics produced 16,135 distinct peptides that validated 4200 transcripts (FDR ≤1%). The catalog of transcripts assembled in this study, together with the identified peptides, dramatically expands and refines planarian gene annotation, demonstrated by validation of several previously unknown transcripts with stem cell-dependent expression patterns. In addition, our robust transcriptome characterization pipeline could be applied to other organisms without genome assembly. All of our data, including homology annotation, are freely available at SmedGD, the S. mediterranea genome database.
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http://dx.doi.org/10.1101/gr.113779.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129261PMC
July 2011

Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori.

Chem Senses 2011 May 10;36(4):335-44. Epub 2011 Jan 10.

Centro Interdipartimentale di Spettrometria di Massa, University of Firenze, Viale G. Pieraccini no. 6, Florence, Italy.

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.
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http://dx.doi.org/10.1093/chemse/bjq137DOI Listing
May 2011

Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor gamma-independent regulation of nucleophosmin.

Hepatology 2010 Aug;52(2):493-505

Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Florence, Italy.

Unlabelled: Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelial cancers, including hepatocellular carcinoma (HCC). The effective anticancer properties and the underlying molecular mechanisms of these drugs in vivo remain unclear. In addition, the primary biological target of TZD, the ligand-dependent transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), is up-regulated in HCC and seems to provide tumor-promoting responses. The aim of our study was to evaluate whether chronic administration of TZD may affect hepatic carcinogenesis in vivo in relation to PPARgamma expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARgamma-expressing and PPARgamma-deficient mouse models of hepatic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes by differential in gel electrophoresis and mass spectrometry analysis. Identified TZD targets were confirmed in cultured PPARgamma-deficient hepatocytes. TZD administration in hepatitis B virus (HBV)-transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in the liver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARgamma deletion in hepatocytes of HBV-transgenic mice (Tg[HBV]CreKOgamma) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARgamma-deficient hepatocytes. TZD inhibited NPM expression at protein and messenger RNA levels and decreased NPM promoter activity. TZD inhibition of NPM was associated with the induction of p53 phosphorylation and p21 expression.

Conclusion: These findings suggest that chronic administration of TZD has anticancer activity in the liver via inhibition of NPM expression and indicate that these drugs might be useful for HCC chemoprevention and treatment.
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http://dx.doi.org/10.1002/hep.23669DOI Listing
August 2010

CSF proteomic analysis in patients with normal pressure hydrocephalus selected for the shunt: CSF biomarkers of response to surgical treatment.

Neurol Sci 2010 Jun 21;31(3):283-91. Epub 2009 Nov 21.

Department of Neurosurgery, University of Florence, Largo P. Palagi, 1, 50139, Florence, Italy.

The aim of our pilot study was to investigate, by a proteomic approach, the expressed differences in cerebrospinal fluid (CSF) protein patterns in order to aid in the diagnosis and treatment of normal pressure hydrocephalus (NPH). Seventeen patients with NPH, selected by Intracranial-Pressure monitoring (ICPmo), underwent implantation of a shunt and after 6 months were clinically re-evaluated. Thirteen patients improved, whereas four did not. During ICPmo CSF was collected and its proteoma was analyzed by 2D gel electrophoresis and mass spectrometry. The over-expression of alpha2HS glycoprotein, alpha1 antichimotrypsin and alpha1beta glycoprotein and the under-expression of glial fibrillary acidic protein, apolipoproteins (AIV, J and E), complement C3c, anti-thrombin, alpha2 antiplasmin and albumin seem to be associated with a positive response to surgery. Most of these proteins have been reported to be altered in Alzheimer disease, supporting the hypothesis of a possible link between these two nosological entities.
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http://dx.doi.org/10.1007/s10072-009-0181-0DOI Listing
June 2010

Solution behaviour and biomolecular interactions of two cytotoxic trans-platinum(II) complexes bearing aliphatic amine ligands.

Chemistry 2009 Sep;15(36):9139-46

Inorganic Chemistry Department, Universidad Autónoma de Madrid, 28045 Madrid, Spain.

A novel trans-platinum(II) complex bearing one dimethylamine (dma) and one methylamine (ma) ligand, namely trans-[PtCl(2)(dma)(ma)], recently synthesised and characterised in our laboratory, displayed relevant antiproliferative properties in vitro, being more active than the parent complex, trans-[PtCl(2)(dma)(ipa)], which has isopropylamine (ipa) in place of methylamine. We have analysed comparatively the solution behaviour of these two complexes under various experimental conditions, and investigated their reactivity with horse heart cytochrome c by mass spectrometry, inductively coupled plasma-optical emission spectroscopy (ICP-OES), 2D [(1)H,(15)N],[(1)H,(13)C] HSQC and [(1)H,(1)H] NOESY NMR. Some important changes that occurred in the [(1)H,(13)C] HSQC NMR spectrum of cytochrome c treated with trans-[PtCl(2)(dma)(ma)] in water, after two days' incubation, most probably arose from direct platinum coordination to the protein side chain; this was proved conclusively by [(1)H,(1)H] NOESY NMR and [(1)H,(15)N] HSQC NMR measurements. Met65 was identified as the primary Pt binding site on cytochrome c. Electrospray mass spectrometry (ESIMS) results provided evidence for extensive platinum-protein adduct formation. A fragment of the [Pt(amine)(amine')] type was established to be primarily responsible for protein metalation. ICP-OES analysis revealed that these trans-platinum(II) complexes bind preferentially to the serum proteins albumin and transferrin rather than to calf thymus DNA. Pt binding to DNA was found to be far lower than in the case of cisplatin. The implications of the results for the mechanism of action of novel cytotoxic trans-platinum complexes are discussed.
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http://dx.doi.org/10.1002/chem.200901090DOI Listing
September 2009

Unambiguous characterization and tissue localization of Pru P 3 peach allergen by electrospray mass spectrometry and MALDI imaging.

J Mass Spectrom 2009 Jun;44(6):891-7

Dipartimento di Chimica Organica e Industriale, Università di Parma, Viale G.P. Usberti 17a, I-43100 Parma, Italy.

The lipid transfer protein (LTP), Pru p 3, has been identified as the major allergen present in peach, and its sequence obtained by direct amino acid sequencing has been previously reported. However, several sequences, obtained from c-DNA and available in databases, show differences among them and from the originally proposed structure. In this paper, we report the fast and unambiguous determination of the structure of Pru p 3 protein, extracted from three different varieties of peach, by electrospray ionization mass spectrometry (ESI-MS), both coupled to single stage (quadrupole) or advanced (FT-HRMS) analyzers. The structure was identical to one of the cDNA-derived sequences and different in two positions from the previously reported structure obtained by amino acid sequencing. Moreover, the exclusive localization of the protein in the outer part of the fruits was assessed by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI MSI). The results reported here demonstrate the full potential of mass spectrometry for rapidly obtaining high quality structural data of relevant food proteins.
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http://dx.doi.org/10.1002/jms.1562DOI Listing
June 2009

MALDI mass spectrometry imaging, from its origins up to today: the state of the art.

Comb Chem High Throughput Screen 2009 Feb;12(2):156-74

Centro Interdipartimentale di Spettrometria di Massa, Universita degli Studi di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.

Mass Spectrometry (MS) has a number of features namely sensitivity, high dynamic range, high resolution, and versatility which make it a very powerful analytical tool for a wide spectrum of applications spanning all the life science fields. Among all the MS techniques, MALDI Imaging mass spectrometry (MALDI MSI) is currently one of the most exciting both for its rapid technological improvements, and for its great potential in high impact bioscience fields. Here, MALDI MSI general principles are described along with technical and instrumental details as well as application examples. Imaging MS instruments and imaging mass spectrometric techniques other than MALDI, are presented along with examples of their use. As well as reporting MSI successes in several bioscience fields, an attempt is made to take stock of what has been achieved so far with this technology and to discuss the analytical and technological advances required for MSI to be applied as a routine technique in clinical diagnostics, clinical monitoring and in drug discovery.
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http://dx.doi.org/10.2174/138620709787315454DOI Listing
February 2009

Detection of honeybee venom in envenomed tissues by direct MALDI MSI.

J Am Soc Mass Spectrom 2009 Jan 7;20(1):112-23. Epub 2008 Sep 7.

Interdepartmental Centre of Mass Spectrometry, University of Florence, Florence, Italy.

A new analytical approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for the study of honeybee venom is shown. In vitro and in vivo models simulating the bee sting have been developed using live honeybees and, as the envenomation sites, pig ears and rat legs; MALDI MSI has been used to map, over time, the diffusion and distribution of three venom allergens (Api m 1, Api m 4, and Api m 6) and two venom toxins (apamine and mast cell degranulating peptide). In conjunction with other classical biochemical techniques and high resolution mass spectrometry (HRMS), structural data have been obtained that contribute to current understanding of honeybee venom composition. Initial data have also been obtained demonstrating the feasibility of mapping the organism's response to the sting. The opportunity to monitor venom diffusion and the organism's response at the same time might open new pathways for in vivo preclinical studies in designing and testing new venom immunotherapy (VIT).
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http://dx.doi.org/10.1016/j.jasms.2008.09.006DOI Listing
January 2009

Exploring proteins in Anopheles gambiae male and female antennae through MALDI mass spectrometry profiling.

PLoS One 2008 Jul 30;3(7):e2822. Epub 2008 Jul 30.

Centro Interdipartimentale di Spettrometria di Massa, Università degli Studi di Firenze, Firenze, Italy.

MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002822PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474704PMC
July 2008